The present invention relates the isolation from human plasma of an endotoxin-inactivator which could be added to pyrogenic lots of blood products, such as albumin, immune globulins, antihemophilic factor (AHF) concentrates, Factor IX concentrates, interferons, fibronectin and others, thus rendering them non-pyrogenic, and again suitable for clinical use.
Pyrogenicity in blood products is caused by contamination from endotoxins of Gram-negative bacteria during the manufacturing process. Because of the ubiquitous nature of bacteria, the control of these physiologically active agents is of utmost importance to the plasma fractionation industry, as well as to the entire pharmaceutical industry. The most positive method of control, strict aseptic techniques that limit microbial contamination, cannot, in most cases, maintain complete sterility throughout the manufacturing process. Therefore, manufacturers may at times find their final products pyrogenic at the bulk solution stage prior to filling. The result may be loss of the entire lot, or only partial recovery.
In the plasma fractionation industry the monetary loss due to pyrogenic products may be measured in millions of dollars per year. However, no plasma fractionator has used or reported any means to depyrogenate their endotoxin-contaminated products except that, in the case of depyrogenation of albumin, there are two documented methods.
The first published method for depyrogenation of clinical albumin was reported by Wye and Kim (Vox Sang 32: 182-184, 1977) who mixed pyrogenic albumin with Cohn ethanol fractions IV-1 and IV-4, based on the findings of Yoshioka and Johnson (J. Immunol. 89: 326-335, 1962), followed by differential thermal heating to recover albumin according to the method of Schneider et al. (Blut 30: 121-134, 1975). The reported method not only requires an excessive amount of Cohn Fractions IV-1 and IV-4, but also suffers considerable losses of albumin. The yield based on 21 batches was about 75%. In some cases, the procedure had to be repeated thus resulting in further losses.
The second method for depyrogenation of albumin was developed by this inventor. It is described in his Ser. No. 635,134, filed July 27, 1984, now abandoned. This method is based on the finding that plasma contains "enzyme(s)" which could detoxify the endotoxins. Pyrogenic albumin was mixed with sufficient plasma to detoxify all endotoxins present. The albumin was then purified to over 99%. (Hao, Vox Sang 36: 313-320, 1979). The resultant albumin had a non-detectable level of endotoxin (less than 0.05 ng/ml) as assayed by Limulus Amebocyte Lysate (LAL) test (Levin et al. J. Lab. Clin. Med. 75: 903, 1970) and confirmed by the USP rabbit pyrogen test.
Obviously, the addition of plasma cannot be used for depyrogenation of other clinical plasma products, such as antihemophilic factor (AHF), immune serum globulin (ISG), Factor IX complex concentrate and plasma protein fraction (PPF), since the resulting product would then contain undesired plasma proteins.
It has been known for over 30 years since the first report by Hegemann (Z. Immunitactsforsch 111: 213-225, 1954) that normal human plasma (serum) has the ability to diminish the pyrogenicity of endotoxin derived from the Gram-negative bacteria. This observation was confirmed in subsequent years by many reports (Skarnes et al., J. Exp. Med. 108: 685-700, 1958; Rall et al., Am. J. Physiol. 188: 559-562, 1957; Rudbach and Johnson, Nature 202: 811-812, 1964; Yoshioka and Johnson, J. Immunol. 89: 326-335, 1962; Landy et al. J. Exp. Med. 110: 731-750, 1959; Skarnes, Ann. N.Y. Acad. Sci. 133: 644-662, 1966). Landy et al. (1959) and Skarnes (1966) further suggested that the detoxifying effect by serum (or plasma) is of an enzymatic nature. Yoshioka and Johnson (1962) fractionated serum by the Cohn ethanol procedure (Cohn et al. JACS 68: 459-475, 1946) and found that Cohn Fraction IV-1 contains the substance(s) which decrease pyrogenicity caused by endotoxins. Skarnes (1966), using plasma fractions obtained from ion exchange chromatography, found that the esterase associated with the alpha-1-lipoprotein appeared responsible for degradation of endotoxin, and an alpha-1-globulin esterase appeared responsible for inactivation of endotoxin.
According to Skarnes, "numerous attempts were made to purify the IV.sub.c fraction in order to separate the active enzymes. However, neither cellulose nor sephadex columns were well suited to the purpose and although subfractions were obtained which were rich in either the lipoprotein esterase or the a.sub.1 -globulin esterase, all such fractions contained both esterases."
Johnson et al. (Amer. J. pathol. 88: 559-574, 1977) later isolated from human serum a single inactivator which was neither a lipoprotein, nor a serine esterase. They did, however, find esterase activity associated with a partially purified inactivator in a sucrose density gradient system, even though, for unknown reasons, they did not proceed to isolate this protein in pure form.
Johnson's inactivator "LPS-1" was isolated by a six-step procedure: fractional precipitation of plasma with ammonium sulfate at 40-60% saturation; gel filtration on Sephadex G-150; anion exchange chromatography on DEAE-cellulose; gel filtration on Sephadex G-200; hydroxylapatite chromatography; and preparative gel electrophoresis. I found that this procedure was too long and too tedious. Even though extreme caution was taken to ensure that every piece of glassware, chemicals, reagents, and every piece of equipment was pyrogen-free, it was very difficult to maintain them pyrogen-free since each step required lengthly dialysis and concentration of the sample. Therefore, many a time a partially purified fraction was found to have lost its activity at some stage during purification. There was no way of knowing whether the loss of activity was due to contamination of endotoxins or denaturation of the inactivator itself. During purification, it became obvious that the original procedure called for so many steps mainly for the removal of a major contaminant, albumin. I found that bulk of the albumin could be removed at the DEAE-Sephadex step if stepwise elution at 0.15M NaCl concentration was used. By the use of Cibacron-Blue Sepharose (CBS) right after DEAE-Sephadex step, I removed the remaining albumin. I used the same buffer system (0.02M phosphate buffer, pH 7.35) in both steps so that the DEAE-eluate could be simply diluted 3-fold or diafiltered prior to application to CBS column. Because of the complete removal of albumin, a highly purified EI could be readily obtained by hydroxylapatite chromatography thus eliminating the steps of gel filtration on G-150 and G-200 and preparative gel electrophoresis (taught by Johnson) which would have been a bottleneck if large quantities of EI were to be prepared.
Endotoxins are high molecular weight complexes, associated with the outer membrane of Gram-negative bacteria that induce pyrogenic reactions upon intravenous administration. Endotoxins contain lipid, carbohydrate and protein. Purified endotoxins do not contain protein, and therefore, are referred to as lipopolysaccharide (LPS). It has been shown that LPS contains three distinct chemical regions; Region I, 0-specific polysaccharide carrying the main serologic specificity, is linked to the core polysaccharide, known as Region II. This core material is linked in turn to the lipid component--Region III or Lipid A. (Westphal, O. Int. Archs. Allergy Appl. Immun. 49: 1-43, 1975 and Bradley, S.G. Ann. Rev. Microbial. 33: 67-94, 1979).
It is the lipid A which is responsible for most, if not all, of the biological activities of endotoxin (Galanos, et al. Eur. J. Biochem. 19: 145-152, 1971; Galanos, et al. Eur. J. Biochem. 22: 218-224, 1971; Luderitz, et al. The chemistry, Biology and Clinical Significance of Endotoxins Univ. of Chicago Press, pp. 9-21, 1973; Rietschel et al. Infect. Immunity 8: 173-177, 1973). For example, when free lipid A is complexed with bovine serum albumin, or human serum albumin, pyrogenicity is induced comparable to that of intact endotoxin according to rabbit pyrogen test (Galanos et al. 1972; and Rietschel et al. 1973). Furthermore, the activity of Lipid A derived from E. coli and various strains of Salmonella are similar and the pattern of febril response is identical to that produced by intact endotoxin (Luderitz et al. 1973).
Lipid A is composed of repeated disaccharides of glucosamine, which is highly substituted with ester-linked long chain fatty acids (Westphal, 1975 and Luderitz et al. Int'l Sympo. on Pyrogen, Univ. College, London pp. 10-19, 1975). Furthermore, it was found that Lipid A is linked to core heteropolysaccharides by 2-keto-3-deoxyoctonic acid (KDO) which is unique to bacterial liposaccharides but does not contribute to endotoxicity (Rietschel et al. 1973). It is the ester-linked fatty acids which are responsible for the biological activity. Removal of fatty acid residue abrogates the biological activity of Lipid A (Westphal, 1975, Bradley 1979, and Luderitz, 1975), but the remainder of the Lipid A molecule may determine solubility, conformation, distribution within the body and affinity for receptor site (Bradley 1979). Based on information described above, it may be concluded that human plasma contains an endotoxin-inactivator, which is most probably an esterase that detoxifies Lipid A by breaking off the ester-linked fatty acids.
The present invention for the isolation of an endotoxin-inactivator from human plasma involves three purification steps:
(1) Adsorption of plasma on DEAE-Sephadex followed by stepwise elution PA0 (2) Adsorption of DEAE eluate on Cibacron-Blue sepharose (CBS) followed by stepwise elution, and PA0 (3) Adsorption of the CBS elute on hydroxylapatite followed by stepwise elution
The final product is homogeneous as judged by polyacrylamide gel electrophoresis (PAGE), sodium-dodecyl sulfate PAGE (SDS-PAGE) and immunoelectrophoresis. The molecular weight of this protein is estimated as falling between 61,000 and 65,000 daltons as judged by SDS-PAGE and high performance liquid chromatography (HPLC).
The purified inactivator has been found to inactivate all three types of lipopolysaccharides tested; they are S. typhosa, S. enteritides and E. coli strain 055: 85. It has also been found that highly pyrogenic albumin can be made non-pyrogenic by mixing it with titrated amount of this inactivator. The inactivation of endotoxin was evidenced by the Limulus amebocyte lysate test in vitro and was further confirmed by the USP rabbit pyrogen test in vivo.
Chibata, U.S. Pat. No. 4,381,239 reviews methods of removing pyrogen: (1) adsorption; (2) decomposition with acid or alkali; (3) decomposition with an oxidizing agent; or (4) filtration. Chibata taught that bacterial LPS is selectively adsorped by a heterocylic nitrogen compound.
The filtration of pyrogens from biological fluids is known. See Hou, U.S. Pat. No. 4,488,969; Grabner, U.S. Pat. No. 3,897,309; Chibata, U.S. Pat. No. 4,160,697; Nakamura, U.S. Pat. No. 4,259,448.
Mannuzza, U.S. Pat. No. 4,380,511 teaches the removal of pyrogens from blood protein products by absorbing the blood protein on blue dextran, washing away the pyrogen with a low ionic strength solution, and desorbing the column with a high ionic strength solution.
Babb, U.S. Pat. No. 4,381,004 speaks of a "microorganism deactivator" for extracorporeal treatment of blood. However, this deactivator is an antimicrobial agent, not an inactivator of endotoxin. Babb provides for adsorption, rather than inactivation, of bacterial endotoxins.
Shanbrom, U.S. Pat. No. 4,069,216 discloses the "reworking" of pyrogenic lots of Factor VIII by a one or two step cold 6% PEG precipitation. The pyrogens are washed away from the Factor VIII precipitate. They are not inactivated. See also Shanbrom U.S. Pat. No. 4,188,318.
Cano, U.S. Pat. No. 4,000,257 teaches use of ethyl or butyl acetate to extract endotoxins from an influenza virus vaccine to obtain a vaccine of low pyrogenicity.
Smith, U.S. Pat. No. 3,659,027 discloses destruction of pyrogens in water intended for parenteral use by strong alkali. Clearly, such harsh treatment is unsuitable for protein preparations.
Dasinger, U.S. Pat. No. 3,644,175 describes the inactivation (by acidification and heating) of endotoxins of gram-negative bacteria intended for use as a protein source.
Akcasu, U.S. Pat. No. 4,070,289 depyrogenates water by distillation under pressure.
GB No. 1,418,286 teaches the removal of pyrogens from urokinase (a product of human urine) by retaining the pyrogens on an anion exchange cellulose, such as diethylamino ethyl (DEAE) cellulose.
GB No. 1,557,545 teaches that urokinase can be reversibly absorbed on a hydrophilic polysaccharide which does not retain pyrogens.